| 翟玉龙 李德洋 杜小红 何显力△ 邢金良.一种新的线粒体基因组DNA捕获探针的制备及初步应用[J].,2016,16(13):2459-2463 |
| 一种新的线粒体基因组DNA捕获探针的制备及初步应用 |
| A New Preparation Method of Mitochondrial DNA Capture Probe and ItsPreliminary Application |
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| DOI： |
| 中文关键词: 基因捕获 线粒体基因组DNA 二代测序 |
| 英文关键词: Gene capture Mitochondria DNA Next-generation sequencing |
| 基金项目:国家自然科学基金项目" 重大国际合作项目"（81320108021） |
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| 中文摘要: |
| 目的：建立新的线粒体基因组DNA杂交捕获探针制备方法并用进行初步应用。方法：通过PCR 技术扩增特异线粒体DNA
片段，并与生物素偶联，最后与标记磁珠的亲和素混合获得捕获探针。并自行制备的线粒体基因组DNA 文库捕获探针与肝癌全
基因组测序文库进行液相杂交。分离捕获产物后PCR 扩增并进行测序分析。结果：成功建立了线粒体基因组杂交捕获探针制备方
法并成功分离线粒体基因组DNA；对测序数据的分析显示：90 %以上测序数据来自线粒体基因组DNA，且覆盖率达到100 %，且
均一性良好。检测到的同质性变异位点数量和异质性变异位点数量与全基因组测序数据产生的结果接近（P=0.9152，P=0.8409）。
结论：新方法制备的线粒体基因组DNA 杂交捕获探针可以从全基因组文库中高效捕获线粒体基因组DNA测序文库。 |
| 英文摘要: |
| Objective:To establish a new method for mitochondrial DNA (mtDNA) capture probe preparation and subsequent apply
this capture probe in next-generation sequencing.Methods:mtDNA was amplified by PCR and subsequently labeled by biotin. The
biotin labeled mtDNA were further connected with Dynabeads via biotin-streptavidin system. Then the mtDNA capture probe were
mixed with the whole genome sequencing libraries developed from HCC tissues. After mtDNA isolation, the mtDNA was amplified and
sequenced with Illumina Hiseq 2000.Results:We successfully established a new low-cost preparation method of mtDNA capture probe
and sequenced the capture product produced by this probe. 90 %sequencing data were derived from mtDNA and covered 100 % bases.
The uniformity of reads was superior to some commercial kits. The amounts of homoplasmic and heteroplasmic variants were near the results
from whole genome sequencing data (P=0.9152, P=0.8409).Conclusion:The capture probe can isolate mtDNA sequencing library
fromwhole genome sequencing library efficiently. |
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